The focus of this proposal is to develop drug candidates that inhibit DNA Adenine Methyltransferase (DAM) as a new class of antibiotics for the treatment of bacterial infection. Widespread antibiotic resistant pathogenic organisms create life-threatening conditions for patients in both hospital and community settings. To deal with increasing drug resistance, new antibacterial agents are desperately needed. We have established purified enzymes and in vitro and in vivo assays for high throughput compound screening, and compound optimization for potency and selectivity. Screening of chemical diversity libraries has identified several new classes of compounds that inhibit bacterial DAM. We will expand the high throughput screening effort initiated in the SBIR Phase I to include assay automation, the screening of additional small molecule template libraries, and the identification and characterization of new lead compounds. Existing lead compounds will be prioritized on the basis of their potential for chemical optimization and mechanism type. Combinatorial and/or focused libraries will be prepared and screened for the most promising template series and structure-activity relationships (SAR) developed. Chemical optimization will be performed with respect to potency, selectivity against mammalian enzymes, in silico and experimental ADME parameters, and uptake by bacterial cells. Potential drug candidates will be evaluated in animal and cell-based model systems for efficacy and toxicity. A wide spectrum of clinically important pathogenic bacteria will be screened for sensitivity to methylation inhibition. The outcome of this 2-year development program will be a drug candidate ready for Phase I clinical evaluation in humans. [unreadable] [unreadable]